Directed evolution of microorganisms

ABSTRACT

The present invention provides methods for directing the evolution of microorganisms comprising the use of mutator genes and growth under conditions of selective pressure. The method discloses mutator genes which can be used in the methods of the present invention and provides ATCC deposits which exemplify the evolved microorganisms produced by the methods.

This is a divisional of application Ser. No. 09/314,847, filed May 19,1999, now U.S Pat. No. 6,365,410.

FIELD OF THE INVENTION

The present invention relates to methods for directing the evolution ofmicroorganisms using mutator genes. Such methods provide a pool ofmicrobial genetic diversity advantageous for industrial application,such as for the industrial production of heterologous proteins, such ashormones, growth factors and enzymes, and the biocatalytic production ofchemicals, vitamins, amino acids and dyes.

BACKGROUND OF THE INVENTION

The industrial applicability of microorganisms is restricted by theirphysiological limits set by solvent, pH, various solutes, salts andtemperature. Organic solvents are generally toxic to microorganisms evenat low concentrations. The toxicity of solvents significantly limits useof microorganisms in industrial biotechnology for production ofspecialty chemicals and for bioremediation applications. Solventmolecules incorporate into bacterial membranes and disrupt membranestructure (Isken and Bont, 1998, Extremophiles 2(3): 229-238); (Pinkartand White, 1997, J. Bacteriol. 179(13): 4219-4226); (Ramos, Duque etal., 1997, “J. Biol. Chem. 272(7): 3887-3890); (Alexandre, Rousseaux etal., 1994, FEMS Microbiol, Lett, 124(1): 17-22); and Kieboom, Dennis etal., 1998, J. of Bacteriology 180(24): 6769-6772). Classic strainimprovement methods including UV and chemical mutagenesis have beenapplied for selection of more tolerant strains (Miller, J., “A ShortCourse In Bacterial Genetics,” Cold Spring Harbor Laboratory Press,1992). A number of studies have been dedicated to identification andisolation of solvent tolerant mutants among various bacterial strains.Spontaneous E. coli solvent tolerant mutants and mutants isolated in theprocess of 1-methyl-3-nitrosoguanidine (NTG) mutagenesis were obtainedfrom strain K-12 (Aono, Albe et al., 1991 Agric. Biol. Chem 55(7):1935-1938). The mutants could grow in the presence of diphenylether,n-hexane, propylbenzene, cyclohexane, n-pentane, p-xylene. VariousPseudomonas strains were able to adapt and to grow in a toluene-watertwo-phase system (Inoue and Horikoshi, 1989, Nature 338: 264-266), withp-xylene (Cruden, Wolfram et al., 1992, Appl. Environ. Microbiol. 58(9):2723-2729), styrene and other organic solvents (Weber, Ooijkaas et al.,1993, Appl. Environ. Microbiol. 59(10): 3502-3504), (de Bont 1998,Trends in Biotechnology 16: 493-499). Yomano et al. isolated ethanoltolerant mutants which increased tolerance from 35 g/l to 50 g/l during32 consequent transfers (Yomano, York et al., 1998, J. Ind. Microbiol.Biotechnol. 20(2): 132-138). High temperature evolution using E. colihas been disclosed in the art (Bennett, 1990, Nature, Vol. 346, 79-81)however the fitness gain was low as compared to the parent.

Strains of E. coli that carry mutations in one of the DNA repairpathways have been described which have a higher random mutation ratethan that of typical wild type strains (see, Miller supra, pp. 193-211).As reported by Degenen and Cox (J. Bacteriol., 1974, Vol. 117, No. 2,pp.477-487), an E. coli strain carrying a mutD5 allele demonstrates from100 to 10,000 times the mutation rate of its wild type parent. Greeneret al., “Strategies In Molecular Biology,” 1994, Vol. 7, pp.32-34,disclosed a mutator strain that produces on average one mutation per2000 bp after growth for about 30 doublings.

Microorganisms are used industrially to produce desired proteins, suchas hormones, growth factors and enzymes and to produce chemicals, suchas glycerol and 1,3 propanediol (WO 98/21340 published May 22, 1998 andU.S. Pat. No. 5,686,276 issued Nov. 11, 1997), vitamins, such asascorbic acid intermediates (1985, Science 230:144-149), amino acids,and dyes, such as indigo (U.S. Pat. No. 4,520,103, issued May 28, 1985).In spite of advances in the art, there remains a need to improve themicroorganisms and methods for producing such desired proteins,chemicals, amino acids and dyes.

SUMMARY OF THE INVENTION

The present invention relates generally to methods for directing theevolution of a microoganism, that is for directing desired geneticchange in a microorganism in response to conditions of selectivepressure. In one aspect, the present invention relates to methods forevolving microorganisms to grow under extreme conditions, such as athigh temperature, under conditions of pH extremes, in the presence ofsolvents, and in the presence of high salt. In another aspect, thepresent invention relates to methods for evolving a microorganismcomprising at least one nucleic acid encoding a desired protein or anenzyme in an enzymatic pathway to grow under desired conditions.

The present invention is based, in part, upon the finding thatmicrorganisms such as wild-type E. coli and E. blattae, can be evolvedinto microorganisms capable of growing in the presence of high solvents,such as DMF and 1,3 propanediol, using methods described herein. Thepresent invention is also based, in part, upon the finding that E. colican be evolved into a microorganism capable of growing at elevatedtemperatures using methods described herein. The present invention isfurther based, in part, upon the identification of the optimal mutationrate for a microorganism and the discovery that the mutation rate can becontrolled.

Accordingly, the present invention provides a method for preparing anevolved microorganism comprising the steps of culturing a microorganismcomprising at least one heterologous mutator gene for at least 20doublings under conditions suitable for selection of an evolvedmicroorganism, wherein said heterologous mutator gene generates amutation rate of at least about 5 fold to about 100,000 fold relative towild type, and restoring said evolved microorganism to a wild typemutation rate. In one embodiment, the microorganism further comprises atleast one introduced nucleic acid encoding a heterologous protein, saidprotein(s) including, but not limited to hormones, enzymes, growthfactors. In another embodiment, the enzyme includes, but is not limitedto hydrolases, such as protease, esterase, lipase, phenol oxidase,permease, amylase, pullulanase, cellulase, glucose isomerase, laccaseand protein disulfide isomerase. The present invention encompassesgenetic changes in the microorganism as well as changes in theintroduced nucleic acid.

In yet a further embodiment, the microorganism further comprisesintroduced nucleic acid encoding at least one enzyme necessary for anenzymatic pathway. In one embodiment, the introduced nucleic acid isheterologous to the microorganism; in another, the introduced nucleicacid is homologous to the microorganism. In a further embodiment, theenzyme is a reductase or a dehydrogenase and said enzymatic pathway isfor the production of ascorbic acid or ascorbic acid intermediates. Inan additional embodiment, the enzyme is glycerol dehydratase or1,3-propanediol dehydrogenase and said enzymatic pathway is for theproduction of 1,3 propanediol, 1,3 propanediol precursors or 1,3propanediol derivatives. In another embodiment, the enzyme isglycerol-3-phosphate dehydrogenase or glycerol-3-phosphate phosphataseand said pathway is for the production of glycerol and glycerolderivatives. In a further embodiment, the enzymatic pathway is for theproduction of amino acids, such as tryptophane or lysine or dyes, suchas indigo.

In one embodiment of the present invention, the microorganism iscultured for between about 20 to about 100 doublings; in anotherembodiment, the microorganism is cultured for between about 100 to about500 doublings; in yet another embodiment, the microorganism is culturedfor between about 500 to about 2000 doublings and in a furtherembodiment, the microorganism is cultured for greater than 2000doublings. In one embodiment, the mutator gene generates a mutation rateof at least about 5 fold to about 10,000 fold relative to wild type; inanother embodiment, the mutator gene generates a mutation rate of aleast about 5 fold to about 1000 fold and in another embodiment, themutator gene generates a mutation rate of about 5 fold to about 100 foldover wild type.

In one embodiment, an evolved microorganism comprises from about 3 toabout 1000 selected mutations in about 3 to about 500 genes and mayfurther comprises from about 20 to about 100,000 neutral mutations. Inone aspect, the mutations generated are non-specific and in anotheraspect, the mutations generated are specific.

In one embodiment of the present invention, the microorganism comprisesa plasmid comprising the heterologous mutator gene and said step ofrestoring said evolved microorganism to a wild type mutation ratecomprises curing the evolved microorganism of said plasmid. In anotherembodiment, the plasmid comprises a temperature sensitive origin ofreplication and the curing comprises growing the evolved microorganismat a restrictive temperature. In a further embodiment, the microorganismcomprises at least one copy of the mutator gene in the chromosome andsaid step of restoring said evolved microorganism to a wild typemutation rate comprises excision or removal of said mutator gene fromthe host genome or the replacement of the mutator gene with a functional(non-mutator) allele of the same gene.

In one embodiment, the present invention comprises the use of at leastone mutator gene to evolve a microorganism. In another embodiment, themutator gene includes but is not limited to a mutD gene mutation, a mutTgene mutation, a mutY gene mutation, a mutM gene mutation, a mutH genemutation, a mutL gene mutation, a mutS gene mutation or a mutU genemutation and homologues of these DNA repair genes which have beenmutated as long as the mutated gene has impaired proofreading function.In a further embodiment, the mutator gene comprises at least one of themutD mutations disclosed herein in Table I.

In one embodiment of the present invention, conditions suitable forselection include but are not limited to culturing said microorganism inthe presence of at least one organic solvent, such as for example,alcohols, diols, hydrocarbon, mineral oil, mineral oil derived products,halogenated compounds and aromatic compounds; in the presence of hightemperature, such as in the range of 42°-48° C.; in the presence of highsalt, and in the presence of extreme pH conditions, such as alkaline oracidic conditions.

The present invention encompasses methods for evolving gram positive andgram negative microorganisms as well as yeast, fungus and eucaryoticcells including hybridomas. In one embodiment, the gram negativemicroorganism includes members of Enterobacteriaceae and in anotherembodiment comprises Eschericia and in another embodiment comprises E.coli and E. blattae. In further embodiments of the present invention,the evolved microorganism includes E. coli having ATCC accession numberPTA-91 and E. blattae having ATCC accession number PTA-92.

The present invention also provides expression vectors and host cellscomprising a mutator gene and methods for producing such vectors andhost cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B shows the nucleic acid (SEQ ID NO:1) and amino acid (SEQ IDNO:2) sequence of the mutD gene. Illustrative examples of mutations ofthe mutD gene are provided.

FIGS. 2A-2D provides the nucleic acid sequences (SEQ ID NO:3 and 5,respectively) for the enzyme 1,3-propanediol dehydrogenase (PDD).

FIGS. 3A-3B provides the amino acid sequences (SEQ ID NO:4 and 6,respectively) for the enzyme 1,3-propanediol dehydrogenase (PDD).

FIG. 4 provides a time course for E. coli cultures subjected to directedevolution and selection under elevated temperature.

FIG. 5—Glycerol fermentation of E. blattae at pH 7.0. Culture conditionsare described in the text. Plate counts were by serial dilution andperformed in triplicate on Luria agar plates. Substrate and Oproductswere measured by HPLC.

FIG. 6—Glycerol fermentation of E. blattae strain GEB031-4 at pH 7.0.Culture conditions are described in the text. Plate counts were byserial dilution and performed in triplicate on Luria agar plates.Substrate and products were measured by HPLC.

DESCRIPTION OF THE MICROORGANISM DEPOSITS MADE UNDER THE BUDAPEST TREATY

Applicants have made the following biological deposits under the termsof the Budapest Treaty on the International Recognition of the Depositof Micro-organisms for the Purposes of Patent Procedure:

Depositor Identification International Reference Depository DesignationDate of Deposit Escherichia coli MM294 ATCC PTA-91 May 17, 1999derivative Escherichia blattae 33429 ATCC PTA-92 May 17, 1999 derivative

DETAILED DESCRIPTION Definitions

A mutation refers to any genetic change that occurs in the nucleic acidof a microorganism and may or may not reflect a phenotypic change withinthe microorganism. A mutation may comprise a single base pair change,deletion or insertion; a mutation may comprise a change, deletion orinsertion in a large number of base pairs; a mutation may also comprisea change in a large region of DNA, such as through duplication orinversion.

When many possible different mutations in nucleic acid can give rise toa particular phenotype, the chance of mutation to that phenotype will behigher than in a situation where only a few types of mutations can giverise to a particular phenotype. As used herein the terms “wild-typemutation” and “spontaneous mutation” are used interchangeably. The rateof spontaneous mutation is defined as the probability of a mutation eachtime the genome is replicated or doubled. As used herein “mutation rate”is simultaneous with “frequency” and refers to the absolute number ofmutations/doubling/base pair. As used herein, the term “relative rate”refers to the ratio of mutation rates of two strains, one of these isusually a wild type strain. Relative rate indicates how much more likelyit is that a strain will undergo mutation as compared to the wild typestrain. The frequency of spontaneous mutation of wild type E. coli (theE. coli genome has about 4.6×10⁶ base pairs) is about 5×10⁻¹⁰ mutationsper base pair per doubling (see Drake, 1991). Doubling refers to theprocess of reproduction of at least part of a genome of an organism andusually involves reproduction by binary fission. As used herein,“doubling” encompasses the reproduction of nucleic acid within anmicroorganism achieved by any means.

As used herein, a “mutator strain” refers to a microorganism having ahigher than naturally occurring rate of spontaneous mutation. As usedherein, “mutator gene” refers to a DNA repair gene which comprises amutation and which has impaired proof reading function. As used herein,the term “mutator plasmid” refers to a plasmid or expression vector orcassette comprising a mutator gene. Culturing a microorganism comprisinga mutator gene will give rise to mutational events during genomereplication. The present invention encompasses the use of any DNA repairgenes comprising mutations as long as the mutated DNA repair gene iscapable of introducing mutational events in a microorganisms genome oron a gene introduced into the microorganism. DNA repair genes includebut are not limited to, mutD, mutT, mutY, mutM, mutH, mutL, mutS or mutUand homologues of these genes. A homologue as used herein refers to afunctionally related DNA repair gene. In one embodiment, the mutatorgene is a mutD gene (the epsilon subunit of DNA polymerase III, seeDegnen et al., 1974, J. Bacteriol. 117:477-487) comprising mutationsthat provide an impaired proofreading function. In one embodimentdisclosed herein, the mutD mutation is introduced into a microorganismon a plasmid. Illustrative embodiments of MutD mutations are disclosedherein in Table I. The mutD mutations impair proofreading function ofthe epsilon subunit of DNA polymerase III holoenzyme by significantdecrease in the 3′-5′ exonuclease activity (Takano et al., 1986, Mol.Gen. Genet. 205(1):9-13).

When referring to mutations or genetic changes in an evolvedmicroorganism, “neutral mutation” refers to a mutation which has littleor no measurable effect on the phenotype of an evolved strain under agiven set of conditions. Examples of “neutral mutations” include, butare not limited to, silent mutations which do not lead to a change inthe amino acid sequence of the encoded protein, mutations which affectproteins that are not essential for growth under a given set of cultureconditions, and mutations in non-coding regions of the chromosome. Inone illustrative embodiment herein, an E. coli strain evolved for hightemperature was characterized as being auxotrophic for three amino acids(ie, were not able to grow in medium without Cys/Met, Asp/Asn, and Pro)indicating that there were at least three neutral mutations in the E.coli in addition to the mutations associated with growth at hightemperature. The term “selected mutation” as used herein refers to thosemutations which are associated with a phenotype of an evolved strainunder a given set of conditions. Being associated with means that themutation is directly or indirectly responsible for the improved oraltered phenotype.

When referring to mutations or genetic changes in a host cell ormicroorganism, nonspecific refers to the changes in the host cell genomewhich occur randomly throughout the genome and which potentially canaffect all bases and includes frameshifts. Non-specific mutationsencompass changes in single base pairs as well as changes in a largenumber of base pairs as well as changes in large regions of DNA. Forexample, in one embodiment, an evolved microorganism which has beenexposed to a MutD gene comprising mutations that impair the proofreading function will comprise random mutations at a rate of about5-1000 times over wild type. In one illustrative embodiment of themethod using a mutD mutation, the evolved strain had at least 3 randommutations. The present invention encompasses any rate of mutations thatprovides the desired phenotype. When referring to genetic changes in ahost cell, specific mutation refers to mutations which can becharacterized or which comprise definable genetic changes, such as A:Tto C:G transversion characteristic of mutT mutations; G:C to T:Atransversion characteristic of mutY and mutM mutations; A:T to G:C andG:C to A:T transitions and frameshifts characteristic of mutH, mutL,mutS, uvrD (mutU) mutations; G:C to T:A transversions characteristic ofthe mutYmutM double mutation (Miller et al., A Short Course in BacterialGenetics, a Laboratory Manual and Handbook for E. coli and RelatedBacteria).

When referring to a mutator gene, “heterologous” means that the gene isintroduced into the cell via recombinant methods and is preferablyintroduced on a plasmid. The mutator gene may also be introduced intothe microorganism genome through recombinant techniques. The mutatorgene introduced into the microorganism may be a mutation of a naturallyoccurring DNA repair gene in the cell or may be foreign to the hostmicroorganism. Referring to nucleic acid as being “introduced” into amicroorganism means that the nucleic acid is put into the microorganismusing standard molecular biology techniques. An introduced nucleic acidmay be the same or different than nucleic acid naturally occurring inthe microorganism.

As used herein the term “restoring to wild type mutation rate” refers tothe process whereby a mutator gene is removed from an evolvedmicroorganism thereby restoring the wild-type mutation rates. Thepresent invention encompasses any process for removing the mutator genefrom an evolved organism and includes but is not limited to curing theorganism of a resident plasmid comprising the mutator gene or byexcising or otherwise removing the mutator gene from the host genomesuch that normal DNA repair function is restored. Curing refers tomethods for producing cells which are free of a plasmid comprising themutator gene. A microorganism can be cured of any resident plasmid usingtechniques known to the skilled artisan.

DETAILED DESCRIPTION

One of the basic tenants of inheritance is that mutations occur randomlyand then are selected by the environment. Mutations that happen toconfer a selective advantage on the organism are preferentially passedon to future generations. The present invention relates to methods fordirecting desired genetic change in a microorganism, ie directing theevolution of a microorganism, by exposing the microorganism to a mutatorgene, selecting for acquisition of desired characteristics in theevolved microorganism, and curing the microorganism of the mutator gene,or otherwise removing the mutator gene, such that wild type mutationrates are restored.

I. Uses of the Invention

In one aspect of the present invention, the methods are used to evolve amicroorganism to grow under extreme conditions, such as in the presenceof elevated temperature, high solvent, altered pH or in the presence ofhigh salt. In another aspect of the present invention, the methods areused to evolve microorganisms which comprise introduced nucleic acidencoding a heterologous protein or at least one enzyme in an enzymatic,ie biocatalytic pathway. Such commercially important proteins includehormones, growth factors and enzymes. Illustrative biocatalytic pathwaysinclude those disclosed in U.S. Pat. No. 5,686,276, issued Nov. 11,1997, for the production of 1,3-propanediol and in 1985, Science230:144-149 for the production of ascorbic acid intermediates.

Methods of the present invention are especially advantageous forproducing improved microorganisms used for the biocatalytic productionof chemicals and vitamins where numerous catalytic events are takingplace either concurrently or sequentially within the host microorganism.In such complex biocatalytic systems, it is often difficult to identifythe specific molecular events causing low yields, host toxicity orcatalytic failures and therefore difficult if not impossible tounderstand which specific genetic events to alter in order to correctthe deficiencies. The methods of the present invention provide theadvantage of allowing the microorganism to make the required changes inresponse to selective pressure.

Additionally, the methods of the present invention provide an advantagefor obtaining microorganisms comprising desired phenotypic traitsassociated with multiple genes, such as the ability of a microorganismto grow at elevated temperatures. The use of the mutator gene provides ameans for producing genetic diversity and the simultaneous growth underconditions of selective pressure allows the microorganism to identifythe specific genetic changes required for survival. The use of mutD genemutations allows for very large diversity to be provided to themicroorganism from which to select for the specific genetic changes thatprovide a growth advantage. Therefore, the methods disclosed hereinavoid the limited diversity that is often produced with art methods thatbegin the directed evolution process with defined sets of genes.Furthermore, the methods disclosed herein eliminate additional screeningsteps often associated with art methods for producing genetic diversity.A further advantage of the present invention is that the methods can beapplied to microorganisms which have not been sequenced and for whichthere may be limited information upon which to design genetic changes.

In illustrative embodiments disclosed herein, a mutated mutD generesiding on a plasmid was introduced via recombinant techniques into E.coli or E. blattae. The E. coli or E. blattae cell was then culturedunder conditions suitable for growth for a time sufficient for at least20 doublings and up to at least about 2000 doublings under conditions ofselective pressure. In one example, E. coli was grown under conditionsof increased temperature or in the presence of DMF and in another E.blattae was growth in the presence of solvent, such as DMF or 1,3propanediol. As a result, E. coli was evolved into a microorganismcapable of growing at temperatures up to about 48° C. or in the presenceof 80 g/l DMF. E. coli evolved to grow at elevated temperatures alsobecame auxotrophic for three amino acids, Cys/Met, Asp/Asn and Pro. E.blattae was evolved into a microorganism capable of growinganaerobically in the presence of at least 105 g/l 1,3-propanediol andwhich comprised genetic changes in at least one catalytic activityassociated with 1,3 propanediol production, 1,3-propanedioldehydrogenase, shown in FIG. 3. (SEQ ID NO:4).

The use of a plasmid comprising a mutator gene, ie, a mutator plasmid,can be used to control the mutation rate of a microorganism. Asdescribed under Section II below, plasmid constructs can be designedwhich provide reduced levels of expression of a mutator gene therebyproviding a means for altering the ratio of naturally occurring DNArepair genes vs mutator genes. This provides a means for combining theadvantage of mutD mutations (which results in random mutagenesis) withthe advantages of the other known mutators (lower mutation frequencywhich leads to a lower burden on the cells). Additionally, plasmidconstructs can be designed that allow for curing the evolvedmicroorganism of the mutator gene, such as the use of a temperaturesensitive origin, thereby allowing for a means for turning the mutationevents off and on in the microorganism. For a gram positivemicroorganism, such as B. subtilis where the entire genome has beensequenced, the present invention could encompass the steps of deletingor mutating a DNA repair gene, evolving the Bacillus, and restoring thenaturally occurring DNA repair system through recombination events. Asdisclosed herein, several members of Escherichia, such as E. coli and E.blattae have been subjected to the directed evolution methods.Illustrative examples of evolved E. coli and E. blattae have beendeposited with the ATCC and have accession numbers PTA-91 and PTA-92,respectively.

The methods of the present invention provide a means to accomplishlong-term evolution of microorganisms. An E. coli strain comprising aplasmid comprising a mutD mutation was grown for >1000 doublings withouta reduction in mutation rate. The present invention also provides ameans for reducing the functional genome of an organism. A microorganismcan be grown for many thousands of generations, such that only the geneswhich are essential would remain functional. Most of the other geneswould carry random and inactivating mutations.

The present invention also provides a means for making non-pathogenicorganisms. A pathogenic strain can be evolved into a mutator strain byintroduction of a mutator gene and grown for extended periods of time.As a result many of the genes that are involved in pathogenicity wouldbecome inactivated and the strain would be safe to use.

The present invention also provides a means to streamline the metabolismof an organism. A strain which has an improved yield on nutrients or areduced metabolic rate (maintenance metabolism) can be produced bymethods disclosed herein. Such strains would be useful productionstrains for chemicals as well as enzymes. The present invention providesa means for making microorganisms mutator strains by introducing amutator gene, thereby protecting the microorganism's naturally occurringDNA repair genes from becoming mutator genes in response to selectivepressure. That is, the introduction of the mutator plasmid into amicroorganism whether via a plasmid or into the genome, protects thecells from developing a mutator phenotype in response to selectivepressure.

II. Mutator Genes and Frequency of Mutations

Mutator genes of the present invention include but are not limited to,mutations of the DNA repair genes mutD, mutT, mutY, mutM, mutH, mutL,mutS or mutU or their homologues in other microorganisms. A descriptionof the DNA repair genes are disclosed in Miller, supra; mutD isdisclosed in Maki et al., 1983, Proc. Natl. Acad. Sci., U.S.A. 80,7137-7141 (GenBank accession number K00985.1 GI: 147678 and FIG. 1); B.subtilis mutS and mutL are disclosed in Ginetti et al., 1996,Microbiology, August, 142 (Pt 8): 2021-9; Streptococcus pneumoniae hex Brepair gene, mutL of Salmonella typhimurium and PMS1 of Saccharomycescerevisiae are disclosed in Prudhomme et al., 1989, J. Bacteriology,October; 171 (10): 5332-8; Streptococcus pneumoniae hexA and mutS ofSalmonella typhimurium and E. coli are disclosed in Priebe et al., J.Bacteriol, 1988, January; 170(1): 190-6 and Prudhomme et al., 1991, J.Bacteriol. November; 173(22): 7196-203; human mutS homologue, hMSH2, andhuman MutL homologue, hMLH1, are disclosed in Macdonald et al., 1998,Heptology, July 28(1):90-7; the mut-1 of Neurospora is disclosed inDillon et al., 1994, Genetics, September 138(1):61-74 and yeasthomologues of mutL and mutS are disclosed in WO 97/15657. The methods ofthe present invention comprises the use of at least one of the mutantDNA repair genes and may involve the use of more than one. It ispreferred that a mutator gene be dominant to the wild type gene of themicroorganism such that mutations are introduced into the genome of themicroorganism. In a preferred embodiment, the mutator gene is a mutationof the mutD gene. The nucleic acid and amino acid sequence for mutD isshown in FIG. 1 (SEQ ID NO:1 and 2, respectively). One particular mutDmutation, mutD5, is disclosed in Takano, K., et al., (1986, Mol GenGenet 205, 9-13, Structure and function of dnaQ and mutD mutators ofEscherichia coli). Strain CSH116 was obtained as disclosed in Miller, J.H. (1992, A Short Course in Bacterial Genetics). This strain is reportedto carry the mutD5 allele. The mutD gene in this strain was found to bevery different from the published mutD5. The mutD gene from strainCSH116 is designated herein as mutD5′. Table I gives the mutations foundin mutD5 and mutD5′. Further mutations in mutD which result in increasedlevels of mutation frequency were identified recently in Taft-Benz, S.A. et al., (1998, Nucl. Acids Res. 26, 4005-4011, Mutational analysis ofthe 3′-5′ proofreading exonuclease of Escherichia coli DNA polymeraseIII). Table I describes various mutD mutations useful in the presentinvention. Table II describes various promoters used with the mutDmutations and Table III describes mutator plasmids and the range ofavailable mutation frequencies in E. coli.

TABLE I mutations in the coding region of mutD MutD Clone #nucleo-#amino amino amino tide acid nucleotide acid nucleotide acid  44  15 CThr T Ile mutD5′ 218  73 T Leu G Trp mutD5 369 123 T Thr C Thr mutD5′418 138 C Pro T Pro mutD5′ 451 151 T Ala C Ala mutD5′ 484 161 G Leu AArg mutD5′ 491 164 C Ala T Val mutD5 661 220 A Glu C Asp mutD5′ 665 222A Ile C Leu mutD5′ 673 225 T Ala A Ala mutD5′ 688 228 C Leu T Leu mutD5′706 236 A Lys G Lys mutD5′ 715 239 T Ser C Ser mutD5′ 727 243 A Arg GArg mutD5′

TABLE II mutD Mutations Name Mutations wild type ATGACCGCTATG... (SEQ IDNO:7) pOS100 TTG A-CGCT TTG... (SEQ ID NO:8) pOS101 GTGACCGCT GTG...(SEQ ID NO:9) pOS102 GTG-CCGCT GTG... (SEQ ID NO:10) pOS104 TTGACCGCTTTG... (SEQ ID NO:11) pOS105 GTGACCGCTGTGAGCACTT(G)CAATTAcACGCCAGATCGTTCTCGATACCGAAAT(C)... (SEQ ID NO:12)pOS106 GTGACCGCT-TG... (SEQ ID NO:13)

TABLE III Mutator (mutD5) and control (mutD) plasmids and the range ofavailable mutation frequencies in E. Coli. mut. Frequency mutator rate #plasmid genotype ori ab resistance size (kb) (average) (relative)  1pMutD5-61 mutD5′ pSc kan 5.97  6.4 × 10⁻⁵ ˜1000-fold  2 pMutD71-Ts mutDpSc kan 5.97  2.5 × 10⁻⁸ wild type  3 pBRmutD68 mutD5′ pBR322 kan, bla6.16  1.1 × 10⁻⁴ (AL data) ˜10000-fold  4 pBRmutD727 mutD pBR322 kan,bla 6.16 nd wild type Modified  5 pOS100 mutD5′ pBR322 kan, bla 6.16   2× 10⁻⁵ ˜800-fold  6 pOS101 mutD5′ pBR322 kan, bla 6.16  3.8 × 10⁻⁶˜152-fold  7 pOS102 mutD5′ pBR322 kan, bla 6.16  1.1 × 10⁻⁶ ˜44-fold  8pOS104 mutD5′ pSc kan 5.97 4.35 × 10⁻⁷ ˜17-fold  9 pOS105 mutD5′ pSc kan5.97  1.1 × 10⁻⁶ ˜44-fold 10 pOS106 mutD5′ pSc kan 5.97   5 × 10⁻⁶˜200-fold

MutD mutations can introduce all types of base pair changes includingframe shifts (Miller, supra). MutD5 has a reported relative mutationfrequency of 1000-10000 fold in rich medium, ie, 5×10⁻⁶ to 5×10⁻⁷mutations per doubling per base pair (Denegen et al., 1974, J.Bacteriol. 117, 477-478). Considering that the E. coli genome has4.6×10⁶ bp, then a mutD5 gene will generate 2.3 to 23 mutations perdoubling per genome. In a preferred embodiment of the present invention,mutator plasmids have been generated which allow for reduced expressionlevels of the mutated mutD repair gene such that the mutation raterelative to wild type is reduced. As illustrated in Table II, the MutDgene has 2 closely located ATG start codons with 6 nucleotides betweenthem. The first ATG is considered to be putative. Both ATG codons werereplaced with GTG or TTG codons for reducing mutD5′ expression levels.The space between the 2 ATG codons up to 5 nucleotides was truncated.The plasmids comprising these mutator genes provide lower mutation rateswhen introduced in E. coli in comparison to MutD5′ plasmids.

As a result, the microorganism comprising the plasmid comprising themutated mutD gene expressed normal levels of the functional epsilonsubunit coded by naturally occurring mutD and low amounts of thenon-functional epsilon subunit coded by the mutated mutD5′. Bothsubunits compete for polymerase III. Consequently, the microorganismwill most of the time have functional proof-reading but to a certainfraction of time the cell will copy its DNA without proof-reading, dueto the presence of the mutD mutations. Thus, the frequency ofmutagenesis of a microorganism can be altered by adjusting theexpression of the mutator gene or by altering the ratio of a naturallyoccurring DNA repair gene to the corresponding mutated DNA repair gene.The mutations caused by these plasmid introduced mutator genes shouldstill be as random as mutations caused by a chromosomal copy of mutD5.Data generated in Example III indicate that mutation rates of 5-1000times over wild type are preferred for most applications. Other means ofcontrolling the mutation frequency include having two copies of mutD onthe plasmid or integrated into the microrganism genome and/or using atransmissible heat sensitive plasmid which could be used to temporarilytransform cells into mutators and then restore them to wild type rates.Yet another way to adjust the mutation frequency is to identify mutDmutations which result in moderate mutation frequency due to reducedproof reading. Such mutants have been recently identified but it is notknown if these mutations preferentially result in a few types ofspecific mutations, Taft-Benz et al., 1998, Nucl. Acids. Res.26:4005-4011. Mutation rates are determined using rifampicin orstreptomycin as disclosed in Horiuchi, et al., 1978, Mol. Gen. Genet.163:227-283.

Mutation rates and a description of the molecular fingerprint of amicroorganism produced by the methods disclosed herein and claimed arealso exemplified by virtue of the microorganism deposits made with theATCC under the terms of the Budapest treaty.

III. Construction of Mutator Genes and Mutator Plasmids

Construction of plasmids comprising mutator genes and transformations ofmicroorganisms can be performed by means deemed to be routine to theskilled artisan. In one embodiment illustrated herein, nucleic acidencoding a mutator gene is introduced into a microorganism on areplicating plasmid, ie, a mutator plasmid, which is cured or otherwiseeliminated from the microorganism after evolution. In another embodimentdisclosed herein, nucleic acid encoding a mutator gene is introducedinto a microorganism's genome in addition to or as a replacement of anaturally occurring DNA repair gene.

Nucleic acid encoding a mutator gene can be isolated from a naturallyoccurring source or chemically synthesized as can nucleic acid encodinga protein or enzyme. Sources for obtaining nucleic acid encoding DNArepair mutD, mutT, mutY, mutM, mutH, mutL, mutS or mutU is provided inSection II. FIG. 1 provides the nucleic acid (SEQ ID NO:1) and aminoacid sequence (SEQ ID NO:2) for mutD and Table I and III providepreferred mutations for mutD and the mutation rates obtained for eachconstruct. Once nucleic acid encoding a mutator gene is obtained,plasmids or other expression vectors comprising the mutator gene may beconstructed using techniques well known in the art. Molecular biologytechniques are disclosed in Sambrook et al., Molecular Biology Cloning:A Laboratory Manual, Second Edition (1989) Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989) and Brown, T. Current Protocolsin Molecular Biology, Supplements 21, 24, 26 and 29. Nucleic acidencoding a mutator gene is obtained and transformed into a host cellusing appropriate vectors. A variety of vectors and transformation andexpression cassettes suitable for the cloning, transformation andexpression in bacteria are known by those of skill in the art.

Typically, the plasmid vector contains sequences directing transcriptionand translation of the nucleic acid, a selectable marker, and sequencesallowing autonomous replication or chromosomal integration. Suitablevectors comprise a region 5′ of the gene which harbors transcriptionalinitiation controls and a region 3′ of the DNA fragment which controlstranscriptional termination. These control regions may be derived fromgenes homologous or heterologous to the host as long as the controlregion selected is able to function in the host cell.

Initiation control regions or promoters, which are useful to driveexpression of the mutator gene. Virtually any promoter capable ofdriving expression is suitable for the present invention. Once suitablecassettes are constructed they are used to transform the host cell.General transformation procedures are taught in Current Protocols InMolecular Biology (Vol. 1, edited by Ausubel et al., John Wiley & Sons,Inc., 1987, Chapter 9) and include calcium phosphate methods,transformation using PEG, electroporation and protoplast transformation.

After subjecting a microorganism to directed evolution using a mutatorplasmid, the microorganism is cured of the mutator plasmid in order torestore the microorganism to wild-type mutation rates. Methods forcuring a microorganism of a resident plasmid comprising a mutator geneinclude transformation of the microorganism comprising a mutator plasmidwith an incompatible plasmid; electroporation techniques as described inHeery et al., 1989, Nucl. Acids. Res., 17: 10131; growth with acridineorange or ethidium bromide in the medium (Jeffrey Miller, 1972, inCuring of Episomes from E. Coli strains with Acridine Orange fromExperiments in Molecular Genetics, Cold Spring Harbor Laboratories, pg.140). In this method, acridine orange is added to 5 ml cultures of anEnterobacteriaceae strain at 125 μg/ml and allowed to grow overnight at37° C. The following day, the cultures are plated out and individualcolonies are used to prepare plasmid nucleic acid. The nucleic acid isanalysed by means known to those of skill in the art to determine thepresence or absence of the plasmid. For microorganisms comprising amutator gene in their genome, techniques known to those of skill in theart can be used to restore the microorganism back to wild-type mutationrates, such as excising the mutator gene or replacing the mutator genewith the naturally occurring DNA repair gene through homologousrecombination techniques.

IV. Culture Conditions and Selective Pressure

Once a microorganism has been exposed to a mutator gene, it is culturedunder conditions of desired selective pressure, such as elevatedtemperature, pH, salt or in the presence of a solvent, such as, forexample, DMF or 1,3 propanediol. Examples of other solvents includealcohols, diols, hydrocarbon, mineral oil, mineral oil derived products,halogenated compounds and aromatic compounds.

As the skilled artisan will appreciate, growth conditions are straindependent. General growth conditions are disclosed in Truesdell et al.,(1991, Journal of Bacteriology, 173: 6651-6656) and Sonoyama et al.(1982, Applied and Environmental Microbiology, Vol. 43, p. 1064-1069).Culture media may be supplemented when selectable markers are presentsuch as antibiotic resistance genes, including but not limited totetracycline, ampicillin or chloramphenicol.

For the methods of the present invention, cultures may be grownaerobically or anaerobically in either liquid medium or solid medium,depending upon the microorganism and the type of selection. If culturesare grown in liquid medium, it is preferred to undergo a number ofrounds of replication (20 or more) in order to allow the survivors ofthe selection to grow over the wild-type. If cultures are grown in solidmedium, such as on an agar plate, it is preferred to have a number ofrepetitive platings in order to pick the survivors directly and to applyhigher selection pressure in each round and to amplify the populationthat is able to grow under the specific conditions of selection.

The manner and method of carrying out the present invention may be morefully understood by those of skill in the art by reference to thefollowing examples, which examples are not intended in any manner tolimit the scope of the present invention or of the claims directedthereto.

EXAMPLES Example 1 Construction of mutD and mutD5′ Plasmids and Testingin 3 Bacterial Strains

The following example illustrates the construction of plasmidscomprising the mutator gene, mutD5′.

mutD and mutD5′ genes were amplified by PCR using mutd1(5′-CGCCTCCAGCGCGACAATAGCGGCCATC-3′ SEQ ID NO:14) and mutd2(5′-CCGACTGAACTACCGCTCCGCGTTGTG-3′ SEQ ID NO:15) primers from genomicDNA of E. coli and E. coli CSH116 (Miller 1992), respectively. The PCRproducts were cloned into pCR-Blunt vector (Invitrogen, Carlsbad,Calif.). Plasmids from clones with the correct orientation were isolatedand digested with Smal-HindIII restriction enzymes. The overhang endswere filled using T4 polymerase and cloned into pMAK705 plasmid digestedwith Smal-PvuII. The ligation products were transformed into JM101competent cells. The resulted plasmids had the temperature-sensitiveorigin of replication, carried kanamycin resistance marker and werenamed pMutD-71 (control plasmid, wild type genotype) and pMutD5-61(mutator plasmid).

The plasmids were successfully tested in E. coli MM294 (F⁻ endA1 hsdR17(r_(k) ⁻ m_(k) ⁺)supE44 thi-1 relA1) and E. blattae ATCC accessionnumber 33429 for evolution of solvent tolerance. All evolutionexperiments were performed in LB medium. Mutation rates were determinedby plating aliquots of cell suspensions on LB plates containing 100ug/ml rifampicin or streptomycin. The mutation frequency was calculatedby dividing the number of resistant cells by the total number of platedcells.

Example 2 Evolution of Solvent Tolerance

The following example illustrates the evolution of solvent tolerantmicroorganisms using the mutator plasmids constructed as in Example 1.

In order to make evolution experiments quantitative, LB agar platessupplemented with 50, 60, 70, 80 and 90 g/l DMF and 25 ug/ml kanamycinwere used. The size of every evolving population was limited to 10⁶cells. After each plating, the number of raised colonies was counted and10 were selected for the next plating. Cells from selected colonies weremixed together and aliquots containing 10⁶ cells were plated on freshplates containing the same and higher concentrations of DMF. After 2consequent platings the cells were cured of the plasmids by growth atelevated temperatures. E. blattae 33429 and E. coli MM294 were cured at41° C. and 43° C., respectively. Three to four subculturings atindicated temperatures were sufficient for 87-100% curing. Individualcured clones were selected by parallel growth of clones in selective andnon-selective medium. The curing was also confirmed by plasmidpurification from selected clones and gel analysis.

The cured strains were tested for growth with the same DMFconcentrations as their plasmid containing parents. The experimentdemonstrates (1) the advantage of strains harboring mutator plasmidsover strains carrying control plasmids and (2) the preservation ofevolved features in strains cured from mutator plasmids.

The results of the short-term evolution are summarized in Table IV. Inthe process of 2 platings we obtained E. coli colonies, which were ableto grow on plates containing 20 g/l higher concentration of DMF thancontrol clones. Analysis of E. coli MM294 harboring control and mutatorplasmids revealed that the mutation frequency of cells carrying controlplasmids was more then 3 orders of magnitude lower in comparison withcells containing mutator pMutD5-61. Our results showed thathypermutation was very beneficial for cell survival at elevated DMFconcentrations (Table V). E. blattae 33429 appeared to be more sensitiveto DMF. Population of 10⁶ cells raised 968 colonies during 2 plating.When 10 bigger colonies were mixed and a new aliquots of 10⁶ cells wereplated on DMF plates supplemented with 70 g/l DMF, more than 1000 tinycolonies grew on the plates. However, these small colonies were notviable after transfer on fresh 70 g/l DMF plates. The mutation frequencyof E. blattae 33429(pMutD5-61) dropped from 4.55×10⁻⁶ to 1.1×10⁻⁷ aftersecond plating on plates containing 60 g/l DMF (Table V). PlasmidMutD5-61 initially provided lower mutation frequency in E. blattae incomparison with E. coli. The E. blattae strain distinctly reducedmutability after cultivation in the presence of DMF. Although E. coliand E. blattae strains belong to the family Enterobacteriaceae, thebehavior of E. coli mutD5 gene product could be somewhat different in E.blattae cell environment. Nevertheless, the benefits of pMutD5 forsurvival of E. blattae on 60 g/l DMF plates were obvious. Control cellscarrying pMutD-71 couldn't grow in the presence of DMF above 50 g/l.

Contrary to E. blattae 33429(pMutD5-61), we did not observed significantadjustments of mutability in E. coli strains. The mutation frequencystayed within the same range at the end of evolution experiment. (TableV).

Single colonies of evolved cultures were used for curing experiments.The curing efficiency was 87-100% with E. blattae 33429 and E. coliMM294. The mutation frequencies of cured clones were similar to wildtype control frequencies, and cured clones preserved their ability togrow at elevated DMF concentration. E. blattae 33429 cured clones grewwith 60 g/l DMF and E. coli MM294 grew with 80 g/l DMF. Initialtolerance of MM294 and E. blattae 33429 was 60 g/l and 50 g/l DMFrespectively. The evolved strains increased their tolerance by 20 g/land 10 g/l DMF, respectively. Therefore, sensitivity to DMF is straindependent.

The advantage of mutator plasmids for evolution in liquid culture wastested as well. Within 4 days of solvent tolerance evolution in liquidmedium supplemented with DMF or ethanol, E. blattae 33429(pMutD5-61)demonstrated growth at higher concentrations of both solvents incomparison with control cultures.

Mutator plasmids can be applied for evolution of bacterial tolerance todifferent solvents, various environmental stress and potentially toxicspecialty chemicals of industrial biotechnology. One advantage of thedirected evolution methods disclosed herein is that the evolution ofmicroorganisms carrying mutator plasmids can be stopped at any time.Mutator plasmids can be cured from evolving strains, and therefore,evolved desired features of the whole strain can be preserved.

TABLE IV Evolution of solvent tolerance. Colony formation by resistantclones on LB plates supplemented with various DMF concentrations.Plating 1 Plating 2 DMF Number Number Strain Genotype (g/l) of colonies*of colonies* MM294(pMutD5-61) Mutator 60 g/l low density high densitylawn lawn MM294(pMutD5-61) Mutator 70 g/l  11 824 MM294(pMutD5-61)Mutator 80 g/l  0  4 MM294(pMutD-71) Wild type 60 g/l  17 low densitylawn MM294(pMutD-71) Wild type 70 g/l  0  0 EB33429(pMutD5-61) Mutator50 g/l low density high density lawn lawn EB33429(pMutD5-61) Mutator 60g/l  0 968 EB33429(pMutD-71) Wild type 50 g/l 793 high density lawnEB33429(pMutD-71) Wild type 60 g/l  0  0 *The number of coloniesrepresents survivors from 10⁶ cells plated on LB-DMF plates.

TABLE V Mutation frequencies of bacteria harboring mutator and controlplasmids. Mutation rate Mutation rate Strain before the evolution DMF(g/l)* after the evolution MM294  9.2 ± 6.5 × 10⁻⁵ 80 g/l   4.7 ± 4 ×10⁻⁵ (pMutD5-61) MM294 4.15 ± 3.4 × 10⁻⁸ 60 g/l 2.9 ± 2.4 × 10⁻⁸(pMutD-71) EB33429 4.55 ± 3.7 × 10⁻⁶ 60 g/l 1.13 ± 0.9 × 10⁻⁷ (pMutD5-61) EB33429  2.6 ± 2.1 × 10⁻⁸ 50 g/l 4.7 ± 3.8 × 10⁻⁸ (pMutD-71)*Single colonies from LB-DMF plates were grown in LB medium to OD A₆₂₀ =0.8-1.2. and plated on LB-Rifampicin or Streptomycin plates at 30° C.The experiments were done in triplicates.

Example 3 Evolution of High Temperature Strains

Example 3 illustrates high temperature evolution under conditions ofcontinuous fermentation in the mode of turbidostat which allows forfermentation wherein the cell density is stabilized. Two independentexperiments were run with the strains: A: W1485 (ATCC12435) (=nonmutator); B: W1485/pBRmutD68 (same strain but comprises mutatorplasmid). Both strains were gown in continuous culture in a turbidostatin LB medium for about 1800 doublings. The temperature was controlled bya computer based on the measured growth rate to maintain a doubling timeof about 1 h. Whenever the culture grew faster the temperature wasraised and vice versa. The time course of both cultures is shown in FIG.4. Initially, the culture started from W1485/pBRmutD68 evolved fasterthan the culture started from W1485. This indicates the advantage of themutator plasmid. However, After about 400 doublings W1485 reached thehigher temperature. We also measured the mutation rate of samples takenfrom both evolution experiments. Table 6 shows that the starting clonesdiffered in their mutation frequencies by a factor of 3000. However,during the evolution experiments the mutation frequencies converged towithin a factor of 2. The experiment illustrates that the rate oftemperature evolution slows down over the course of the experiment. Itcan be expected that in a wild type strain there are a small number ofgenes which initially limit growth at elevated temperature. Favorablemutations in these genes will lead to relatively large gains in fitness.However, with increasing temperature more and more genes can be expectedto limit growth and the pace of evolution slows down. If individualmutations result in only very small growth benefits to their carrierthen the populations have to be grown for a significant number ofdoublings to enrich the clones carrying these mutations from thepopulation. As a consequence the optimum mutation rate for evolutionwill decrease during the process of evolution. For Table VI, mutationrates were determined using rifampicin as given in Miller (1992, supra).

TABLE VI Mutation rates of strains and populations used for temperatureevolution Strain/population doublings temperature ° C. mutation rateW1485/pBRmutD68 0 45 3142.9 AL018 210 47.22 3428.6 AL019 376 47.502028.6 AL038 1811 48.21 1142.9 W1485 0 45 1.0 AL017 231 47.10 0.9 AL035543 47.91 134.3 AL040 1385 48.57 514.3

Example 4 Directed Evolution of E. blattae and Selection in the Presenceof a Solvent, 1,3-propanediol

E. Blattae ATCC accession number 33429 was transformed with plasmidpMutD68 (see Table III) and cultured in media containing 1, 5, 10, 20,and 30 g/l 1,3 propanediol (cultures are designated as GEBxxx where“xxx” indicates the number of transfers into fresh media). All directedevolution experiments were carried out under anaerobic conditions indefined minimal medium with glycerol as a sole carbon source. E. blattaedoesn't require vitamin B₁₂ for growth, nevertheless, initialexperiments were performed in 2 conditions (1) with B₁₂, and (2) withoutB₁₂ in the growth medium.

Within 18-22 h GEB001 reached maximum 1030-1060 mOD (A₆₅₀) at allconcentrations of 1,3 propanediol. Therefore, 30 g/l 1,3-PD was notinhibitory for GEB001 growth. Growth rates of GEB in the presence of 50g/l were ˜½ of growth rates in the presence of 30 g/l 1,3-PD (590 mOD:1030 mOD in 22 h). The threshold of tolerance to 1,3-PD was foundbetween 70 to 80 g/l. After 10 transfers, GEB010 was able to grow in thepresence of 80 g/l 1,3-PD to 350 mOD within 78 h. However, these cellsfailed to grow at 80 g/l 1,3-PD concentration after next transfer.

E. blattae is known in the art to carry the enzymatic pathway for theproduction of 1,3 propanediol (Roth, et al., 1986, Annu. Rev. Microbiol.50:137-181). In order to determine if E. blattae can make1,3-propanediol in addition to the concentrations of 1,3 propanedioladded to the medium, GEB011 was grown in medium supplemented with 2-¹³Cglycerol and 70 g/l 1,3-PD. The supernatant was then analyzed by NMR(¹³C) and the results indicated the formation of ˜2.6 g/l ¹³C 1,3-PD.Therefore, GEB cells can make 1,3 propanediol in the presence of 1,3-PD.

The evolution of 1,3-propanediol resistance was faster in the presenceof B12. After 2 months of evolution GEB025 (+B12) was able to grow with95-100 g/l 1,3-propanediol. After 3 months of anaerobic growth underselection in the presence of 1,3-propanediol, GEB028 (−B12) could growin medium supplemented with 110 g/l 1,3-propanediol. Analysis of aerobicgrowth of GEB031 on LB plates supplemented with 85, 95, 105 and 115 g/l1,3-propanediol showed that cells produce much bigger colonies in thepresence of 85 g/l in comparison with 105 g/l. No growth was observed at115 g/l 1,3 propanediol. The results indicate that after 3 months ofapplying directed evolutions techniques described herein to E. blattae,the tolerance to 1,3 propanediol was increased from 75 g/l to at least105 g/l under aerobic conditions. The plasmid was cured from the GEB031strain by growing at 41.5 degrees. An illustrative clone, GEB031-4 wasdeposited with the ATCC and has accession number PTA-92 .

Example 5 Genetic Changes in Evolved E. blattae

1,3-propanediol dehydrogenase (PDD) was compared between wild type E.blattae and the evolved strain GEB031. The PDD from the evolved strainhad a higher Km for 1,3-propanediol.

Materials and Methods

Strains—Wild type ATCC 33429, E. blattae comprising the mutant PDD asdescribed in Example 4 and having ATCC accession number PTA-92.

Growth—Cells were grown in a complex medium at 30C 500 ml in a 2800 mlfernbach with shaking at 225 rpm for 20 hr. The medium consists ofKH2PO4, 5.4 g/L; (NH4)2SO4, 1.2 g/L; MgSO47H2O, 0.4 g/L; yeast extract,2.0 g/L; tryptone, 2.0 g/L; and glycerol, 9.2 g/L in tap water. The pHwas adjusted to 7.1 with KOH before autoclaving (Honda, et al., 1980, J.Bacteriol, 143:1458-1465).

Extract prep—Cells were harvested by centrifugation with care to avoidanaerobic conditions. Pellets were resuspended in 100 mM Tricine pH 8.2containing 50 mM KCl and 1 mM DTT. Cells were disrupted by passagethrough a French pressure cell. Crude extracts were clarified bycentrifugation at 20K×g for 20 min followed by 100K×g for 1 hr to yieldthe high speed supernatant (HSS) fraction.

Assays—the assay for PDD was performed as described by Johnson, E. A. etal., 1987, J. Bacteriol. 169:2050-2054.

Partial purification of PDD—HSS was separated on a 16×100 Poros 20 HQcolumn. The buffers were A, 50 mM HEPES, pH 7.4 containing 100 uM MnCland B, A buffer containing 500 mM KCl. The column was loaded anddeveloped at 10 ml/min. The gradient was 10 CV wash, a linear gradientto 70% B in 10 CV, and 1 CV to 100% B. The activity was detected in thevery early fractions of the gradient. Pooled column fractions of the33429 strain were used as collected for assays after the addition ofadditional of DTT to 1 mM. The active fractions from strain GEB031 werepooled and concentrated on a PM30 membrane and used as concentratedafter the addition of additional 1 mM DTT.

Strain GD (U/mg) PDD (U/mg) Ratio GD/PDD 33429 0.64 0.22 2.9 GEB031 0.790.08 9.9

PDD Kinetics—The results are shown below.

PDD Kinetics-The results are shown below. Strain Km (mM Propanediol) Km(uM NAD) 33429 28 57

Example 6 Cloning and Sequencing the 1,3-propanediol Dehydrogenase Genes(dha T) From E. blattae.

The dhaT genes were amplified by PCR from genomic DNA from E. blattae astemplate DNA using synthetic primers (primer 1 and primer 2) based onthe K. pneumoniae dha T sequence and incorporating an Xbal site at the5′ end and a BamHl site at the 3′ end. The product was subcloned intopCR-Blunt II-TOPO (Invitrogen). The cloning dha T were then sequencedwas standard techniques.

The results of the DNA sequencing are given in SEQ ID NO:3 and SEQ IDNO:4.

Primer 1 (SEQ ID NO:14) 5′ TCTGATACGGGATCCTCAGAATGCCTGGCGGAAAAT3′ Primer2 (SEQ ID NO:15) 5′ GCGCCGTCTAGAATTATGAGCTATCGTATGTTTGATTATCTG3′

As will be readily understood by the skilled artisan, nucleic acidsequence generated via PCR methods may comprise inadvertent errors. Thepresent invention also encompasses nucleic acid encoding PDD obtainablefrom E. blattae having ATCC accession number PTA-92.

Example 7 Comparison of Wild-type E. blattae (ATCC Accession Number33429) and the Evolved Strain GEB031-4 (ATCC Accession Number PTA-92).

This example shows that E. blattae subjected to the methods of thepresent invention and having ATCC accession number PTA-92 can completelyconsume 800 mM glycerol during anaerobic fermentation and does notaccumulate 3-hydroxy-propionaldehyde (3HPA) and does not lose viability.In contrast, the wild-type E. blattae accumulates 50 mM 3 HPA andbecomes non viable after consuming only 350 mM glycerol.

The wild-type E. blattae and the evolved E. blattae were subjected tofermentation in the following medium: 75 g glycerol, 5 g K₂HPO₄.3H₂O, 3g KH₂PO₄, 2 g (NH₄)₂SO₄, 0.4 g MgSO₄.7H₂O, 0.2 g CaCl₂.2H₂O, 4 mgCoCl₂.2H₂O, 2 g yeast extract, and 1 g peptone per liter water. The pHwas maintained with 20% NaOH. Both fermentations were run at 30° C. witha N₂ sparge and were inoculated with a stationary grown overnightpreculture.

All references cited herein, including patents, patent applications,sequences and publications are hereby incorporated in their entirety byreference.

17 1 741 DNA Escherichia coli 1 atgaccgcta tgagcactgc aattacacgccagatcgttc tcgataccga aaccaccggt 60 atgaaccaga ttggtgcgca ctatgaaggccacaagatca ttgagattgg tgccgttgaa 120 gtggtgaacc gtcgcctgac gggcaataacttccatgttt atctcaaacc cgatcggctg 180 gtggatccgg aagcctttgg cgtacatggtattgccgatg aatttttgct cgataagccc 240 acgtttgccg aagtagccga tgagttcatggactatattc gcggcgcgga gttggtgatc 300 cataacgcag cgttcgatat cggctttatggactacgagt tttcgttgct taagcgcgat 360 attccgaaga ccaatacttt ctgtaaggtcaccgatagcc ttgcggtggc gaggaaaatg 420 tttcccggta agcgcaacag cctcgatgcgttatgtgctc gctacgaaat agataacagt 480 aaacgaacgc tgcacggggc attactcgatgcccagatcc ttgcggaagt ttatctggcg 540 atgaccggtg gtcaaacgtc gatggcttttgcgatggaag gagagacaca acagcaacaa 600 ggtgaagcaa caattcagcg cattgtacgtcaggcaagta agttacgcgt tgtttttgcg 660 acagatgaag agattgcagc tcatgaagcccgtctcgatc tggtgcagaa gaaaggcgga 720 agttgcctct ggcgagcata a 741 2 246PRT Escherichia coli 2 Met Thr Ala Met Ser Thr Ala Ile Thr Arg Gln IleVal Leu Asp Thr 1 5 10 15 Glu Thr Thr Gly Met Asn Gln Ile Gly Ala HisTyr Glu Gly His Lys 20 25 30 Ile Ile Glu Ile Gly Ala Val Glu Val Val AsnArg Arg Leu Thr Gly 35 40 45 Asn Asn Phe His Val Tyr Leu Lys Pro Asp ArgLeu Val Asp Pro Glu 50 55 60 Ala Phe Gly Val His Gly Ile Ala Asp Glu PheLeu Leu Asp Lys Pro 65 70 75 80 Thr Phe Ala Glu Val Ala Asp Glu Phe MetAsp Tyr Ile Arg Gly Ala 85 90 95 Glu Leu Val Ile His Asn Ala Ala Phe AspIle Gly Phe Met Asp Tyr 100 105 110 Glu Phe Ser Leu Leu Lys Arg Asp IlePro Lys Thr Asn Thr Phe Cys 115 120 125 Lys Val Thr Asp Ser Leu Ala ValAla Arg Lys Met Phe Pro Gly Lys 130 135 140 Arg Asn Ser Leu Asp Ala LeuCys Ala Arg Tyr Glu Ile Asp Asn Ser 145 150 155 160 Lys Arg Thr Leu HisGly Ala Leu Leu Asp Ala Gln Ile Leu Ala Glu 165 170 175 Val Tyr Leu AlaMet Thr Gly Gly Gln Thr Ser Met Ala Phe Ala Met 180 185 190 Glu Gly GluThr Gln Gln Gln Gln Gly Glu Ala Thr Ile Gln Arg Ile 195 200 205 Val ArgGln Ala Ser Lys Leu Arg Val Val Phe Ala Thr Asp Glu Glu 210 215 220 IleAla Ala His Glu Ala Arg Leu Asp Leu Val Gln Lys Lys Gly Gly 225 230 235240 Ser Cys Leu Trp Arg Ala 245 3 1164 DNA Escherichia blattae 3atgagctatc gtatgtttga ttatctggtt ccaaatgtga acttctttgg cccgggcgcc 60gtttctgttg ttggccagcg ctgccagctg ctggggggta aaaaagccct gctggtgacc 120gataagggcc tgcgcgccat taaagacggt gctgtcgatc agaccgtgaa gcacctgaaa 180gccgccggta ttgaggtggt cattttcgac ggggtcgagc cgaacccgaa agacaccaac 240gtgctcgacg gcctggccat gttccgtaaa gagcagtgcg acatgataat caccgtcggc 300ggcggcagcc cgcacgactg cggtaaaggc attggtattg cggccaccca cccgggtgat 360ctgtacagct atgccggtat cgaaacactc accaacccgc tgccgcccat tattgcggtc 420aacaccaccg ccgggaccgc cagcgaagtc acccgccact gcgtgctgac taacaccaaa 480accaaagtaa aatttgtgat tgtcagctgg cgcaacctgc cttccgtctc cattaacgat 540ccgctgctga tgatcggcaa gcccgccggg ctgaccgccg ccaccggtat ggatgccctg 600acccacgcgg tagaggccta tatctccaaa gacgccaacc cggttaccga tgcctctgct 660attcaggcca tcaaactgat tgccaccaac ttgcgccagg ccgtcgccct ggggaccaac 720ctcaaagccc gtgaaaacat ggcctgcgcc tctctgctgg ccgggatggc ctttaacaac 780gccaacctgg gctatgttca cgccatggct caccagctgg gcggcctgta cgacatggcc 840cacggggtgg cgaacgcggt cctgctgccc catgtctgcc gctataacct gattgccaac 900ccggaaaaat ttgccgatat cgccaccttt atgggggaaa acaccaccgg tctttccacc 960atggacgcag cggagctggc catcagcgcc attgcccgtc tgtctaaaga tgtcgggatc 1020ccgcagcacc tgcgtgaact gggggtaaaa gaggccgact tcccgtacat ggcagaaatg 1080gccctgaaag acggcaacgc cttctctaac ccgcgcaaag ggaacgaaaa agagattgcc 1140gacattttcc gccaggcatt ctga 1164 4 387 PRT Escherichia blattae 4 Met SerTyr Arg Met Phe Asp Tyr Leu Val Pro Asn Val Asn Phe Phe 1 5 10 15 GlyPro Gly Ala Val Ser Val Val Gly Gln Arg Cys Gln Leu Leu Gly 20 25 30 GlyLys Lys Ala Leu Leu Val Thr Asp Lys Gly Leu Arg Ala Ile Lys 35 40 45 AspGly Ala Val Asp Gln Thr Val Lys His Leu Lys Ala Ala Gly Ile 50 55 60 GluVal Val Ile Phe Asp Gly Val Glu Pro Asn Pro Lys Asp Thr Asn 65 70 75 80Val Leu Asp Gly Leu Ala Met Phe Arg Lys Glu Gln Cys Asp Met Ile 85 90 95Ile Thr Val Gly Gly Gly Ser Pro His Asp Cys Gly Lys Gly Ile Gly 100 105110 Ile Ala Ala Thr His Pro Gly Asp Leu Tyr Ser Tyr Ala Gly Ile Glu 115120 125 Thr Leu Thr Asn Pro Leu Pro Pro Ile Ile Ala Val Asn Thr Thr Ala130 135 140 Gly Thr Ala Ser Glu Val Thr Arg His Cys Val Leu Thr Asn ThrLys 145 150 155 160 Thr Lys Val Lys Phe Val Ile Val Ser Trp Arg Asn LeuPro Ser Val 165 170 175 Ser Ile Asn Asp Pro Leu Leu Met Ile Gly Lys ProAla Gly Leu Thr 180 185 190 Ala Ala Thr Gly Met Asp Ala Leu Thr His AlaVal Glu Ala Tyr Ile 195 200 205 Ser Lys Asp Ala Asn Pro Val Thr Asp AlaSer Ala Ile Gln Ala Ile 210 215 220 Lys Leu Ile Ala Thr Asn Leu Arg GlnAla Val Ala Leu Gly Thr Asn 225 230 235 240 Leu Lys Ala Arg Glu Asn MetAla Cys Ala Ser Leu Leu Ala Gly Met 245 250 255 Ala Phe Asn Asn Ala AsnLeu Gly Tyr Val His Ala Met Ala His Gln 260 265 270 Leu Gly Gly Leu TyrAsp Met Ala His Gly Val Ala Asn Ala Val Leu 275 280 285 Leu Pro His ValCys Arg Tyr Asn Leu Ile Ala Asn Pro Glu Lys Phe 290 295 300 Ala Asp IleAla Thr Phe Met Gly Glu Asn Thr Thr Gly Leu Ser Thr 305 310 315 320 MetAsp Ala Ala Glu Leu Ala Ile Ser Ala Ile Ala Arg Leu Ser Lys 325 330 335Asp Val Gly Ile Pro Gln His Leu Arg Glu Leu Gly Val Lys Glu Ala 340 345350 Asp Phe Pro Tyr Met Ala Glu Met Ala Leu Lys Asp Gly Asn Ala Phe 355360 365 Ser Asn Pro Arg Lys Gly Asn Glu Lys Glu Ile Ala Asp Ile Phe Arg370 375 380 Gln Ala Phe 385 5 1164 DNA Escherichia blattae 5 atgagctatcgtatgtttga ttatctggtt ccaaatgtra acttctttgg cccgggcgcc 60 gtttctgttgttggccagcg ctgccagctg ctggggggta aaaaagccct gctggtgacc 120 gataagggcctgcgcgccat taaagacggt gctgtcgatc agaccgtgaa gcacctgaaa 180 gccgccggtattgaggtggt cattttcgac ggggtcgagc cgaacccgaa agacaccaac 240 gtgctcgacggcctggccat gttccgtaaa gagcagtgcg acatgataat caccgtcggc 300 ggcggcagcccgctcgactg cggtaaaggc attggtattg cggccaccca cccgggtgat 360 ctgtacagctatgccggtat cgaaacactc accaacccgc tgccgcccat tattgcggtc 420 aacaccaccgccgggaccgc cagcgaagtc acccgccact gcgtgctgac taacaccaaa 480 accaaagtaaaatttgtgat tgtcagctgg cgcaacctgc cttccgtctc cattaacgat 540 ccgctgctgatgatcggcaa gcccgccggg ctgaccgccg ccaccggtat ggatgccctg 600 acccacgcggtagaggccta tatctccaaa gacgccaacc cggttaccga tgcctctgct 660 attcaggccatcaaactgat tgccaccaac ttgcgccagg ccgtcgccct ggggaccaac 720 ctcaaagcccgtgaaaacat ggcctgcgcc tctctgctgg ccgggatggc ctttaacaac 780 gccaacctgggctatgttca cgccatggct caccagctgg gcggcctgta cgacatggcc 840 cacggggtggcgaacgcggt cctgctgccc catgtctgcc gctataacct gattgccaac 900 ccggaaaaatttgccgatat cgccaccttt atgggggaaa acaccaccgg tctttccacc 960 atggacgcagcggagctggc catcagcgcc attgcccgtc tgtctaaaga tgtcgggatc 1020 ccgcagcacctgcgtgaact gggggtaaaa gaggccgact tcccgtacat ggcagaaatg 1080 gccctgaaagacggcaacgc cttctctaac ccgcgcaaag ggaacgaaaa agagattgcc 1140 gacattttccgccaggcatt ctga 1164 6 387 PRT Escherichia blattae 6 Met Ser Tyr Arg MetPhe Asp Tyr Leu Val Pro Asn Val Asn Phe Phe 1 5 10 15 Gly Pro Gly AlaVal Ser Val Val Gly Gln Arg Cys Gln Leu Leu Gly 20 25 30 Gly Lys Lys AlaLeu Leu Val Thr Asp Lys Gly Leu Arg Ala Ile Lys 35 40 45 Asp Gly Ala ValAsp Gln Thr Val Lys His Leu Lys Ala Ala Gly Ile 50 55 60 Glu Val Val IlePhe Asp Gly Val Glu Pro Asn Pro Lys Asp Thr Asn 65 70 75 80 Val Leu AspGly Leu Ala Met Phe Arg Lys Glu Gln Cys Asp Met Ile 85 90 95 Ile Thr ValGly Gly Gly Ser Pro Leu Asp Cys Gly Lys Gly Ile Gly 100 105 110 Ile AlaAla Thr His Pro Gly Asp Leu Tyr Ser Tyr Ala Gly Ile Glu 115 120 125 ThrLeu Thr Asn Pro Leu Pro Pro Ile Ile Ala Val Asn Thr Thr Ala 130 135 140Gly Thr Ala Ser Glu Val Thr Arg His Cys Val Leu Thr Asn Thr Lys 145 150155 160 Thr Lys Val Lys Phe Val Ile Val Ser Trp Arg Asn Leu Pro Ser Val165 170 175 Ser Ile Asn Asp Pro Leu Leu Met Ile Gly Lys Pro Ala Gly LeuThr 180 185 190 Ala Ala Thr Gly Met Asp Ala Leu Thr His Ala Val Glu AlaTyr Ile 195 200 205 Ser Lys Asp Ala Asn Pro Val Thr Asp Ala Ser Ala IleGln Ala Ile 210 215 220 Lys Leu Ile Ala Thr Asn Leu Arg Gln Ala Val AlaLeu Gly Thr Asn 225 230 235 240 Leu Lys Ala Arg Glu Asn Met Ala Cys AlaSer Leu Leu Ala Gly Met 245 250 255 Ala Phe Asn Asn Ala Asn Leu Gly TyrVal His Ala Met Ala His Gln 260 265 270 Leu Gly Gly Leu Tyr Asp Met AlaHis Gly Val Ala Asn Ala Val Leu 275 280 285 Leu Pro His Val Cys Arg TyrAsn Leu Ile Ala Asn Pro Glu Lys Phe 290 295 300 Ala Asp Ile Ala Thr PheMet Gly Glu Asn Thr Thr Gly Leu Ser Thr 305 310 315 320 Met Asp Ala AlaGlu Leu Ala Ile Ser Ala Ile Ala Arg Leu Ser Lys 325 330 335 Asp Val GlyIle Pro Gln His Leu Arg Glu Leu Gly Val Lys Glu Ala 340 345 350 Asp PhePro Tyr Met Ala Glu Met Ala Leu Lys Asp Gly Asn Ala Phe 355 360 365 SerAsn Pro Arg Lys Gly Asn Glu Lys Glu Ile Ala Asp Ile Phe Arg 370 375 380Gln Ala Phe 385 7 12 DNA Artificial Sequence wild type mutD gene 7atgaccgcta tg 12 8 11 DNA Artificial Sequence pOS100 mutD mutated gene 8ttgacgcttt g 11 9 12 DNA Artificial Sequence pOS101 mutD mutated gene 9gtgaccgctg tg 12 10 11 DNA Artificial Sequence pOS102 mutD mutated gene10 gtgccgctgt g 11 11 12 DNA Artificial Sequence pOS104 mutD mutatedgene 11 ttgaccgctt tg 12 12 55 DNA Artificial Sequence pOS105 mutDmutated gene 12 gtgaccgctg tgagcacttg caattacacg ccagatcgtt ctcgataccgaaatc 55 13 11 DNA Artificial Sequence pOS106 mutD mutated gene 13gtgaccgctt g 11 14 28 DNA Artificial Sequence primer 14 cgcctccagcgcgacaatag cggccatc 28 15 27 DNA Artificial Sequence primer 15ccgactgaac taccgctccg cgttgtg 27 16 36 DNA Artificial Sequence primer 16tctgatacgg gatcctcaga atgcctggcg gaaaat 36 17 42 DNA Artificial Sequenceprimer 17 gcgccgtcta gaattatgag ctatcgtatg tttgattatc tg 42

We claim:
 1. A method for preparing an evolved microorganism comprisingthe steps of: a) obtaining a microorganism comprising at least oneheterologous mutator gene and at least one introduced nucleic acidencoding at least one heterologous protein, wherein said at least oneheterologous protein is an enzyme; b) culturing said microorganism forat least 20 doublings under conditions suitable for selection of anevolved microorganism, wherein said heterologous mutator gene generatesa mutation rate at least 5-100,000 fold relative to wild type; and c)restoring said evolved microorganism to a wild type mutation rate. 2.The method of claim 1, wherein said at least one heterologous protein isa hydrolase.
 3. The method of claim 2, wherein said hydrolase isselected from the group consisting of proteases, esterases, lipases,phenol, oxidase, permeases, amylases, pullufanaces, cellulases, glucoseisomerase, laccases, and protein disulfide isomerases.
 4. The method ofclaim 1, wherein said mocroorganism comprises at least one copy of saidmutator gene in its chromosome and said step of restoring said evolvedmicroorganism to wild-type mutation rate comprises excision of siadmutator gene.
 5. The method of claim 4, wherein said mutator genecomprises mutD mutations.
 6. The method of claim 4, wherein said mutatorgene comprises mutD mtutations selected form the group of mutD mutationsset forth in table
 1. 7. A method for preparing an evolved microorganismcomprising the step of: a) obtaining a microorganism comprising at leastone heterologous mutator gene and at least one introduced nucleic acidencoding at least one heterologous protein, wherein said at least oneheterologous protein is an enzyme necessary for an enzymatic pathway; b)culturing said microorganism for at least 20 doublings under conditionssuitable for selection of an evolved microorganism, wherein saidheterologous mutator gene generates a mutation rate of at least5-100,000 fold relative to wild type; and c) restoring said evolvedmicroorganism to a wild type mutation rate.
 8. The method of claim 7,wherein said enzyme is selected from the group consisting of reductasesand dehydrogenases, and further wherein said enzymatic pathway resultsin the production of at least one compound selected from the groupconsisting of ascorbic acid or ascorbic acid intermediates.
 9. Themethod of claim 7, wherein said enzyme is selected from the groupconsisting of glycerol dehydratase and 1,3-propanediol dehydrogenase,and further wherein said enzymatic pathway results in the production ofat least one compound selected from the group consisting of1,3-propanediol precursors, and 1,3-propanadiol derivatives.
 10. Themethod of claim 7, wherein said enzyme is selected from the groupconsisting of glycerol-3-phosphate dehydrogenase andglycerol-3-phosphate phosphatases, and further wherein said enzymaticpathway results in the production of at least one compound selected fromthe group consisting of glycerol and glycerol derivatives.